Project Summary/Abstract Type 1 Diabetes (T1D) is caused by pathogenic autoreactive T cells that recognize and destroy the islet tissue. However, the presence of T cells reactive to self-antigens is not sufficient for disease to occur as autoreactive T cells can be found in healthy control subjects; thus various means are available that control unwanted responses. One of the most important mechanisms is the activity of regulatory T cells (Tregs), which arise both in thymus and in the peripheral immune system as a consequence of exposure to antigens. Defects in Treg numbers, phenotype, and/or function have been described in T1D and other autoimmune diseases. Because immunosuppression by Tregs specific for a limited number of Ags is dominant and can efficiently thwart a polyclonal autoreactive response, Tregs are an attractive target for antigen-specific tolerogenic therapies. However, the specificity and repertoire of Tregs recognizing self-Ags in humans and mice and whether they are different in individuals with autoimmunity is unknown. Recent studies have uncovered unique aspects of self-peptides presentation to autoreactive T cells by MHC class II alleles predisposing to autoimmunity. These peculiarities potentially circumvent negative selection of autoreactive T cells in the thymus and lead to autoreactivity in the target tissue. Given the well-known skewing of the tTreg repertoire towards self-reactivity, biochemical complexities may affect the development and repertoire of Tregs and their ability to recognize self-Ags in the targeted tissues ? suggesting that the Ag specificity of local Tregs will be instrumental in controlling autoimmunity in the tissue. Thus, identifying the Ag specificity of Tregs, especially at the site of inflammation, is critical to improve our understanding of Treg deficiencies in T1D. In this project, we will address this question by examining the overall hypothesis that the biochemical intricacies that promote effector immunity may paradoxically alter Treg efficacy and contribute to the overall failure of Tregs to control autoreactive Tconv cells in autoimmune diseases such as T1D. We propose the following Specific Aims to address this question: 1: Characterization of Treg specificity against known I-Ag7-restricted autoantigens 2: Antigen focused approach to define the specificity of Tregs in T1D patients. 3: Identification of novel ligands for Tregs in NOD mice and T1D patients. It is recognized that altered Treg immunoregulation is an inherent factor in autoimmunity but defining specific Treg defects in T1D patients has been challenging. Thus, defining the specificity and repertoire of Tregs and their reactive TCRs would be an important step towards the discovery of selective Treg defects in T1D and greatly improve our understanding of the immunopathology of disease. Moreover, it would pave the way for therapeutic opportunities aimed at restoring proper Treg immunoregulation in pancreatic islets.